Advances in autogenous dentin matrix graft as a promising biomaterial for guided bone regeneration in maxillofacial region: A review

Autogenous dentin matrix (ADM), derived from a patient’s extracted tooth, can be repurposed as an autologous grafting material in reconstructive dentistry. Extracted teeth provide a source for ADM, which distinguishes itself with its low rejection rate, osteoinductive capabilities and ease of preparation. Consequently, it presents a viable alternative to autogenous bone. Animal studies have substantiated its effective osteoinductive properties, while its clinical applications encompass post-extraction site preservation, maxillary sinus floor augmentation, and guided bone tissue regeneration. Nevertheless, the long-term efficacy of ADM applied in bone regeneration remains underexplored and there is a lack of standardization in the preparation processes. This paper comprehensively explores the composition, mechanisms underlying osteoinductivity, preparation methods, and clinical applications of ADM with the aim of establishing a fundamental reference for future studies on this subject.


Introduction
The field of oral implantology has emerged as a reliable and well-documented technique for effectively treating edentulism, offering long-term functional and esthetic benefits. [1]owever, edentulism frequently leads to the loss of alveolar ridge, thereby complicating periodontal and implant surgeries, restorative treatments, as well as orthodontic procedures. [2]o address this issue, various bone grafting materials are employed for the restoration or enhancement of the alveolar ridge bone.Autogenous bone grafts, considered as the gold standard, provide osteogenesis, osteoinduction, and osteoconduction capabilities.However, their utilization is constrained by complications associated with donor sites, potential additional trauma risks, and limited availability of harvested bone quantities. [3,4]lternative grafting materials, such as allogeneic, xenogeneic, and biological substitutes, have emerged as potential alternatives to autologous bone grafts; however, they present challenges including increased infection risks, unpredictable bone resorption rates, prolonged healing durations, and higher associated costs. [5]Bone graft and substitute materials currently used in the dental field have been classified and expounded (as shown in Table 1).Based on the unique characteristics of various bone graft materials, they are utilized to treat specific types of bone defects.23] The autogenous dentin matrix (ADM), derived from discarded teeth, exhibits compositional similarities with alveolar bones and effectively addresses various limitations associated with other grafting materials. [24]The use of ADM grafts eliminates the potential risks associated with allografts and xenografts, such as cross-contamination, immunogenicity, and donor variability.27][28] The ADM encompasses a multitude of growth factors that are indispensable for osteogenesis, demonstrating both osteoconductive and osteoinductive capabilities. [29,30]Following a series of procedures including thorough cleaning, dehydration, demineralization, and sterilization, autologous dentin can be finely ground to the desired dimensions and effectively utilized for clinical bone augmentation. [31]Currently, its application in bone augmentation techniques is both successful and advancing, positioning it as a commendable alternative to autogenous bone grafts. [32,33]While the suitability of a graft material often hinges on its intended application, this review delves into the histological composition, mechanisms underlying osteoinductivity, preparation process, and clinical applications of ADM (as shown in Table 2), aiming to lay a foundational reference for future ADM studies.

Organic components
The organic component of dentin is predominantly composed of type I collagen fibers, accounting for approximately 90%. [41]hese fibers play an important role in calcification.The trimeric superhelical collagen structure facilitates the deposition of mineralized crystals, attachment of biological factors, and effectively supports bone regeneration by positively influencing osteoblastic cell responses. [18]In addition to collagen, dentin comprises non-collagenous proteins (NCPs), proteoglycans, carbohydrate, lipids, etc. [42] NCPs include phosphoproteins and Non-phosphoproteins.
Non-phosphoproteins.This group comprises osteocalcin, a calcium-binding protein, and crucial growth factors such as transforming growth factor (TGF), bone morphogenetic proteins (BMPs), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and other significant growth factors.[48] Both TGF-β1 and BMPs are capable of independently activating the RunX2 pathway to induce osteogenesis. [49,50]TGF-β1 also functions as a synergistic signaling molecule in conjunction with other growth factors, such as FGF and IGF. [51,52]The signaling networks, which play a crucial role in the migration of mesenchymal stem cells (MSCs) and macrophages to the wound site, also induce significant MSCs and macrophage migration to promote wound healing.Additionally, these networks stimulate MSC proliferation and enhance collagen-like bone matrix production.Other synergistic signaling pathways were observed in ADM.Moreover, TGF-1 was identified as the most abundant growth factor, surpassing BMP-2, FGF, PDGF, and VEGF by more than twofold.Notably, VEGF is recognized as a pivotal mitogen that regulates neo endothelial cell outgrowth for hemotransfusion,    and the pro-angiogenic effects of VEGF were potentiated by the synergistic actions of PDGF and FGF. [53,54]Avery et al also highlighted the significance of considering the synergistic impact of growth factors on bone formation in ADM. [54]It is crucial to acknowledge that these growth factors not only effectively promote the proliferation of new endothelial cells but also stimulate their osteogenic effects in a synergistic manner.Furthermore, it is essential to emphasize that other matrix proteins, such as disaccharide chain proteoglycans, may act as supplementary adjuvants to enhance bioactivity. [55]Moreover, it has been demonstrated that disaccharide chain proteoglycans possess the ability to directly stimulate bone formation by activating both the BMP/TGF-β and classical Wnt/β-catenin signaling pathways, rendering them a potential therapeutic approach for addressing bone-related disorders. [56]

Inorganic mineralized fractions
The inorganic components of dentin primarily consist of apatite crystals, accounting for 70-75%. [57]The crystal structure of tooth roots exhibits a low-crystallinity phosphate structure, which is characteristic of autogenous bone.It has been suggested that the smaller-sized hydroxyapatite (HA) may enhance biodegradation due to its increased solubility. [58]The X-ray diffraction (XRD) analysis conducted by Kim et al [39,59] demonstrated that the crystalline structure, domain size, and Ca/P ion solubility of ADM were comparable to those of autogenous bone calcium phosphate.Various compositions were identified, including HA (Ca/P = 1.75), tricalcium phosphate (TCP) (Ca/P = 1.46), amorphous calcium phosphate (ACP, Ca/P = 1.32), and octacalcium phosphate (OCP, Ca/P = 1.24).Priya et al [60] emphasized that acid-etching dissolution of calcium phosphate complexes leads to the liberation of calcium and phosphorus ions, thereby catalyzing the reprecipitation of apatite on the surface.The proposed dissolution-reprecipitation sequence is suggested to underlie the formation of apatite, thereby potentially enhancing osseointegration in bioceramic composites.Furthermore, the authors observed the emergence of macroporous regions in calcium phosphate composites due to expansive dissolution triggered by the a-TCP and CaO phases.Such macroporosity and surface roughness are believed to promote biological cell proliferation and bone growth. [61]lectron microscopic observations reveal that autogenous dental bone graft material exhibits a densely packed microporous structure with low crystallinity.As a result, the density, roughness, and uniformity of autogenous dentin closely resemble those of autogenous cortical bone.Moreover, the collagen network reinforces the blood clot within the alveolar socket, serving as a robust biomechanical scaffold facilitating the migration of osteoblastic cells, it also serves as a reservoir for bone-enhancement proteins such as growth factors, thereby ensuring optimal rates of resorption and remodeling in the bone formation process. [62,63]

Osteogenic mechanism
Upon preopening the bone marrow cavity, stem cells and osteoblasts originating from the bone endosteum become exposed. [64]The newly formed socket is filled with plateletrich blood, leading to the subsequent encapsulation of the biomaterial's surface and gaps by blood clots.During the initial phase, platelet degranulation releases various growth factors such as PDGF-AA/AB/BB, TGF-β1, TGF-β2, VEGF,

Alveolar ridge preservation
Elfana et al [34] 2021  and epidermal growth factor (EGF).Additionally, hyaluronan is released while fibronectin from coagulating plasma in the blood deposits on the surface of the biomaterial. [65]The deposition establishes a connection between the biomaterial and the surface of the bone wall, facilitating interaction.The growth factors released through platelet degranulation stimulate various cells including bone marrow cells, endothelial cells, and osteoblasts present in the endosteum of the bone.These factors promote cell migration, differentiation, angiogenesis, and mitosis. [66]uring cell division, a process of "creeping substitution" occurs, wherein daughter cells propel forward while the parent osteoblasts undergo maturation.These mature osteoblasts secrete osteoid and gradually differentiate into bone cells. [67]The continuous occurrence of cell division and creeping substitution ultimately leads to the closure of the gap between the bone wall and the biomaterial. [68]he crux of bone tissue generation lies in the intricate interplay between cells, matrix, and environmental factors, leading to cellular expansion and secretion of matrix molecules. [65,66]n ideal biomaterial for this purpose should exhibit progressive resorption and remodeling capabilities. [69]When considering autogenous dentin as a potential biomaterial for tissue engineering and regenerative medicine, several crucial factors come to the forefront:

Osteoconductivity
The demineralization process of body dentin reveals a crosslinked collagen fiber network, leading to an expansion in the diameter of dentin tubules and the acquisition of appropriate porosity necessary for their function as scaffolds. [70]These optimized scaffolds possess the ability to accommodate cells, coordinate their activities, and present microstructures that facilitate the attachment of cell adhesion molecules derived from blood and platelets. [71]Furthermore, they provide a matrix conducive to recruiting, proliferating, and differentiating bone progenitor cells even prior to resorption. [72]Moreover, autologous dentin matrix can serve not only as a scaffold but also as a carrier for external cells, growth factors, and genes; thus enhancing its versatility and significance in regenerative medicine. [73,74]In a study conducted by Lee et al, [75] the osteogenic potential of demineralized human dentin matrix was evaluated through quantification of MG-63 cell line proliferation and differentiation, in comparison to a composite material consisting of inorganic bovine bone and collagen.Throughout all observed time intervals, cell adhesion and proliferation on the ADM surface were significantly more abundant than those on Bio-Oss collagen (Geistlich, Wolhusen, Switzerland).ADM consistently outperformed Bio-Oss collagen in terms of both cell adhesion and growth at each examined interval.Cells cultured on ADM exhibited not only a more flattened morphology but also displayed a uniform distribution pattern.Insights obtained from confocal laser scanning microscopy emphasized ADM's superior capability for cytoplasmic spreading attachment.Furthermore, immunofluorescence assays revealed notably higher fluorescence intensity of osteocalcin, an essential biomarker for cell differentiation, on ADM, highlighting its exceptional osteoconductivity. [76]

Osteoinductivity
Osteoinductivity is the inherent capacity of a substance to stimulate progenitor cells to differentiate into osteoblasts, which are responsible for bone formation.ADM exemplifies this potential, primarily due to its inclusion of osteogenesisrelated growth factors. [77]These factors stimulate osteogenic precursor cells in the host's connective tissues, directing them towards differentiation and subsequent bone generation. [78]storical research highlights the osteoinductive ability of ADM.Bessho et al [79] successfully isolated bone morphogenetic proteins (BMPs) from human dentin matrix.Although there are differences between BMPs derived from human dentin and human bone, their in vivo functionality remains comparable as both promote similar outcomes in terms of bone formation. [79]Wang and Ike's work further emphasized the osteoinductive efficacy of materials derived from human dentin. [80,81]he study suggests that even small fragments of wisdom teeth, typically considered as surgical waste, may be repurposed as bone graft materials due to the inherent osteoinductive properties of dentin. [82]Additionally, noteworthy observations were made by Kim et al [83][84][85] when transplanting human ADM into the muscular tissue of nude mice.The subsequent emergence of cartilage and bone, along with the observation of newly formed osteoid on ADM granules, attested to its potential in osteoinduction.Nevertheless, Rijal et al [86] expressed a contrasting viewpoint by arguing that the osteoinductive potential of human dentin might not be as evident as suggested by others.However, they acknowledged the non-antigenic properties of ADM which implies that even if its osteoinductive properties are contested, its biocompatibility remains an advantageous feature.
In conclusion, although there exists substantial evidence supporting the osteoinductive capability of ADM, further comprehensive research and controlled studies are imperative to establish a consensus within the scientific community.This debate also underscores the intricate and multifaceted nature of biomaterial research in the field of regenerative medicine.

Biocompatibility
The dentin and bones of the maxilla and mandible both originate from neural crest cells, indicating a shared embryological heritage that results in notably similar compositions.Autologous dentin, when subjected to dehydration, degreasing, and demineralization, exhibits minimal immunogenicity and antigenicity. [79]Moreover, it demonstrates excellent biodegradability and degrades synchronously with new bone formation. [87]Notably, it plays a pivotal role in expediting BMPs release which transforms undifferentiated stromal cells into osteoblasts and odontoblasts while championing bone tissue regeneration -encapsulating the 3 core principles of osteogenesis. [88]he biocompatibility of ADM, combined with its osteoconductive, osteoinductive, and osteogenic properties, renders it a promising alternative to conventional bone graft materials.With further research and elucidation of its benefits, this material holds the potential to supplant bone grafting practices in the dental field.

Process of preparation of ADM grafts
The transformation of discarded teeth into a biocompatible graft material necessitates a multi-step, meticulous process to ensure both safety and efficacy (as shown in Fig. 1).

Cleaning and pretreatment
Discarded teeth undergo an initial cleansing phase, during which all contaminants and extraneous elements are eliminated.This includes: • Extraction of the pulp.
• Removal of calculus, surface granulation tissue, and any prior dental restorations or root canal filling materials.• Thorough rinsing with distilled water to clean off impurities.www.md-journal.com

Chemical and physical processing
Following the cleaning process, various procedures are conducted on the teeth to optimize their compatibility and prolong their shelf-life: [89] • Dehydration: removes water content to stabilize the dentin.
• Degreasing: rids the dentin of any residual lipids or fats.
• Demineralization: by reducing the mineral content of the tooth, dentin becomes more malleable and biologically active, thereby enhancing its malleability and biological activity.This process leads to the enlargement of dentin tubules, which in turn exposes the underlying collagen network.The augmented exposure ensures prompt release of osteoinductive growth factors, thereby amplifying the graft's potential for osteogenesis. [90]The osteogenic profile of demineralized dentin is notably superior to that of non-mineralized dentin.• Calcination: this step entails subjecting the material to controlled thermal conditions, potentially leading to sterilization and modification of its physical properties.• Lyophilization (freeze drying): the moisture is effectively removed from the dentin, ensuring its long-term preservation without any degradation.• Ethylene oxide sterilization: a method to ensure the complete eradication of all potential pathogens, thereby ensuring the safety of the graft for implantation.

Storage
The processed and ground graft can be readily utilized in a clinical setting.Alternatively, it can be stored in a specialized solution.Under appropriate storage conditions, the shelf-life of ADM products can exceed 5 years at room temperature. [91]nsidering their crucial involvement in cellular processes such as migration, adhesion, and differentiation, the majority of preparation procedures are devised to preserve dentin matrix proteins. [92]Although there exist techniques for complete removal of the organic matrix, their osteogenic potential is compromised due to the possible depletion of these valuable proteins. [93]The primary objective is to maintain the biological potency of the graft while ensuring its safety and ease of use.The chairside preparation of ADM involves a meticulous process that carefully balances the dual goals of graft efficacy and safety.The resulting product is a versatile graft material that harnesses the inherent properties of dentin to facilitate bone regeneration. [91]

Demineralization reagents
Demineralization of dentin plays a pivotal role in its preparation as a graft material for diverse clinical applications.Varied demineralization reagents exert distinct effects on the organic and inorganic matrices of dentin. [94,95]4.1.Chelating agents.
• Ethylenediaminetetraacetic acid (EDTA): A widely employed chelating agent in dentistry, particularly in endodontics for the purpose of removing smear layer.When utilized as a demineralizing agent, it selectively binds to calcium ions, resulting in the elimination of hydroxyapatite, the primary mineral constituent of dentin.The notable advantage associated with EDTA lies in its specificity; it predominantly targets the inorganic component while largely preserving the organic matrix.Consequently, this yields a collagen-rich scaffold that fosters favorable cellular adhesion and proliferation. [96]gure 1.Overview of preparation of ADM grafts.ADM = autogenous dentin matrix.

Strong acids.
• 0.6N-HCL (hydrochloric acid): a potent acid capable of effectively demineralizing dentin.However, the utilization of strong acids necessitates precise regulation of exposure duration and concentration to prevent excessive harm to the organic matrix. [97] 2% HNO 3 (nitric acid): Similar to HCL, nitric acid exhibits strong demineralization properties.However, careful control is necessary to prevent potential denaturation of the collagen component while effectively removing mineral content. [72]4.3.Weak acids.
• Acetic acid: being a weak acid, exhibits a milder demineralization effect compared to strong acids.It enables the creation of a graded demineralization effect, facilitating controlled removal of the mineral phase while minimizing impact on the organic matrix. [98] Anthranilic acid: the demineralizing effects and impacts on the organic matrix of this weak acid are comparatively less aggressive when compared to strong acids. [99]e extent of demineralization and potential alteration of the organic matrix is influenced by the concentration, duration of exposure, and specific properties of the chosen reagent. [34,100]iven the absence of a universally accepted protocol for autogenous dentin demineralization, both clinicians and researchers often rely on empirical evidence and tailor their choice of demineralizing solution to suit the specific requirements of their procedures. [100]The primary objective of the demineralization process should be to generate a scaffold that maintains its structural integrity while promoting cellular infiltration, adhesion, and subsequent osteogenesis. [89]n conclusion, the meticulous selection of a demineralization agent, considering its potency and duration of application, plays a pivotal role in determining the quality and effectiveness of demineralized dentin for grafting purposes.When appropriately processed, demineralized dentin can exhibit exceptional osteoconductive properties along with potential osteoinductive characteristics, establishing it as an indispensable tool in bone regenerative treatments. [101]

Demineralization time
An extended demineralization process can result in the degradation of crucial proteins and growth factors associated with osteogenesis.Moreover, it may compromise the structural integrity of the dentin collagen fiber network, leading to increased graft resorption after implantation and impeding successful bone formation. [63]Koga T's investigation on dentin from a healthy population provided valuable insights into the extent of demineralization. [102]At postoperative weeks 4 and 8, both groups exhibited some new bone formation based on microscopic CT and histological observations; however, the partially demineralized group demonstrated a greater extent of new bone formation.This finding suggests that complete demineralization may cause significant damage to essential components of the organic matrix required for new bone creation.In contrast, partial demineralization effectively eliminated most inorganic and immunogenic components while retaining crucial growth factors and bone-forming proteins. [18]As a result, the bone-forming efficacy of demineralized teeth significantly improved.Undemineralized dentin exhibited higher resorption postimplantation compared to its partially demineralized counterpart.Although both types could induce some degree of new bone development, electron microscopic observations revealed that osteoblasts were unable to attach to undemineralized dentin due to insufficient exposure of the collagen fiber network, which serves as an osteoconductive scaffold for osteoblast attachment.The unexposed collagen fiber network and smaller diameter of dentin tubules limited the release of growth factors associated with osteogenesis. [81]The balance between bone resorption and osteogenesis rates was found to be superior in partially demineralized dentin, particularly when its particle size was 1000 μm. [102]n conclusion, demineralization plays a pivotal role in the release of biologically active components.The extent and duration of demineralization are crucial factors in dentin preparation for grafting.Achieving partial demineralization, which strikes a balance between preserving essential growth factors and proteins while enhancing the osteoconductive properties of the graft material, appears to be the most effective approach for bone regeneration.

Alveolar ridge preservation
Following tooth extraction, the absence of periodontal ligaments commonly leads to resorption of the alveolar bone and thinning of the labial bone plate.The inevitable occurrence of alveolar ridge contraction post-extraction has been extensively documented in literature, highlighting its clinical significance. [103]n a recent randomized controlled clinical trial, Elfana et al compared both radiographic and histologic outcomes of autogenous whole-tooth grafts with autogenous demineralized dentin grafts for alveolar ridge preservation following tooth extraction in human subjects.The study confirmed the effectiveness of autogenous demineralized dentin grafts in maintaining volume, demonstrating positive results in histologic/histomorphometric analyses and reporting a low incidence of complications. [34,104]ased on other reports, ADM also demonstrates comparable primary stability to those utilizing xenograft granules. [105]08][109]

Maxillary sinus floor augmentation
In 2003, Murata [108] reported the first clinical case of using an ADM graft in a sinus procedure.Subsequent follow-up studies confirmed exceptional bone formation.Ge's research employed autogenous dental bone powder to address distal mesial bone defects in the posterior maxillary teeth. [74]The findings underscored the treatment's safety and efficacy in rectifying periodontal anomalies.When utilizing the same powder for maxillary sinus floor augmentation, a significant fusion of the boundary between the base of the maxillary sinus and alveolar bone was observed.The seamless integration of autogenous dental bone powder particles with newly formed bone within the sinus occurred.Bone growth initiated from the alveolar region, gradually ascending towards the sinus.112][113]

Implantation
The autogenous tooth bone graft, obtained chairside from extracted teeth, has been successfully utilized for immediate implant placement in fresh sockets with labial bone defects. [36,114]he gaps between the implant and the labial bone wall, as well as any defects in the labial bone, were filled with acellular dermal matrix (ADM) powder to ensure sufficient support of buccal bone.Wang et al [33] conducted a comparative study utilizing autogenous dentin powder and Bio-Oss bone powder.The objective of the study was to address the buccal side gap of the implant and subsequently evaluate changes in bone volume at 6-month intervals.The findings revealed no significant differences in marginal bone loss between the 2 groups at both time points examined.However, patient feedback highlighted a notable advantage of using autogenous dentin powder, as it resulted in reduced postoperative pain and swelling compared to the allograft bone group.Importantly, patients expressed equal satisfaction with the outcomes regardless of the graft material used.These results suggested that while both autogenous dentin powder and Bio-Oss bone powder exhibit similar osteogenic potential in immediate implant scenarios, the former may offer superior postoperative patient comfort.

Guided bone regeneration (GBR)
Extracted teeth affected by severe periodontitis, yet retaining a relatively intact dental hard tissue structure, present an opportunity for grafting.In a clinical trial conducted by Li from 2015 to 2017 involving 40 patients, the effectiveness of autogenous demineralized dentin graft (DDM) was compared to Bio-Oss for immediate implantation combined with GBR. [37]Remarkably, even in sites with post-extraction periodontitis, DDM demonstrated comparable clinical and radiographic outcomes to traditional osseous powder when utilized for immediate implant placement. [115]Besides, ADM grafts, initially introduced for GBR, demonstrated successful maintenance of the formed corticocancellous bone with a dental implant over an average 5-year follow-up. [116]

Combination with other materials
The utilization of a combination of bone tissue regeneration materials often leads to superior outcomes in bone augmentation compared to the use of a single material alone.Initially, autogenous dentin was combined with calcium sulfate plaster. [117]Subsequently, it was further integrated with additional materials such as calcium phosphate ceramics, hydroxyapatite/β -tricalcium phosphate (HA/β-TCP), platelet-rich fibrin (PRF), [118] Bio-Oss, [119] and autogenous bone.ADM combined with recombinant human bone morphogenetic protein-2 has been shown to promote effective bone regeneration without resulting in complications in human subjects. [120]When autogenous dentin bone powder was combined with PRF in tooth extraction sockets to facilitate the osseous regeneration process, a gradual resorption of the dentin particles accompanied by concurrent new bone formation within the augmented area was observed.Postoperatively, patients reported minimal discomfort, and consistent stability of subsequent implant placements was noted. [38]In addition to its autonomous potential, the efficacy of autogenous non-demineralized dentin in alveolar bone grafting can be enhanced through co-administration with MSCs. [121]utogenous tooth bone graft remains a versatile and efficacious grafting material in various clinical scenarios, owing to its capacity for synergistic integration with other materials, thereby offering patients enhanced post-operative comfort and consistent outcomes. [122]Autogenous tooth bone graft prepared chairside is as effective as other bone grafting materials. [6]Further research endeavors are expected to augment our comprehension of optimal application protocols.

ADM has many advantages:
• Ease of acquisition: dental procedures often produce dental waste which can be repurposed.• Cost-effectiveness: dentin offers a cost-effective alternative to many other grafting materials.
• Minimally invasive: harvesting dentin is often less invasive than obtaining autologous bone.• Biosafety: due to its acellular nature, there is a reduced risk of disease transmission and antigenic reactions.• Osteoinductive potential: partially demineralized dentin showcases promising osteoinductive and osteoconductive abilities.
However, there is no universally applicable approach to its preparation.Moreover, ADM may not always be the optimal choice, particularly in cases of extensive bone loss or absence of available teeth for extraction.The selection of an ideal bone graft material should be customized based on the unique characteristics of each specific bone defect to maximize repair outcomes.It is crucial to conduct comprehensive long-term studies in order to substantiate the osteogenic efficacy of ADM.

Table
The main characteristics of various bone grafts.

Table 2
Clinical research reports of ADM.